title: Unsynchronised subunit motion in single trimeric sodium-coupled aspartate transporters.
abstract: Excitatory amino acid transporters (EAATs) are secondary transport proteins that mediate the uptake of glutamate and other amino acids. EAATs fulfil an important role in neuronal signal transmission by clearing the excitatory neurotransmitters from the synaptic cleft after depolarization of the postsynaptic neuron. An intensively studied model system for understanding the transport mechanism of EAATs is the archaeal aspartate transporter GltPh. Each subunit in the homotrimeric GltPh supports the coupled translocation of one aspartate molecule and three Na(+) ions as well as an uncoupled flux of Cl(-) ions. Recent crystal structures of GltPh revealed three possible conformations for the subunits, but it is unclear whether the motions of individual subunits are coordinated to support transport. Here, we report the direct observation of conformational dynamics in individual GltPh trimers embedded in the membrane by applying single-molecule fluorescence resonance energy transfer (FRET). By analysing the transporters in a lipid bilayer instead of commonly used detergent micelles, we achieve conditions that approximate the physiologically relevant ones. From the kinetics of FRET level transitions we conclude that the three GltPh subunits undergo conformational changes stochastically and independently of each other.
authors.affiliation: University of Groningen, Zernike Institute for Advanced Materials, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Guus B
authors.initials: GB
authors.lastname: Erkens
authors.forename: Inga
authors.initials: I
authors.lastname: Hänelt
authors.forename: Joris M H
authors.initials: JM
authors.lastname: Goudsmits
authors.forename: Dirk Jan
authors.initials: DJ
authors.lastname: Slotboom
authors.forename: Antoine M
authors.initials: AM
authors.lastname: van Oijen
mesh_terms: D001224:Aspartic Acid / Q000737:chemistry* / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D050491:Glutamate Plasma Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D008051:Lipid Bilayers / Q000378:metabolism; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D043902:Pyrococcus horikoshii / Q000737:chemistry / Q000378:metabolism; D012964:Sodium / Q000737:chemistry*
chemical_list: D050491:Glutamate Plasma Membrane Transport Proteins; D008051:Lipid Bilayers; D001224:Aspartic Acid; D012964:Sodium